Double stranded RNA induces potent and specific gene silencing in a broad range of eukaryotic organisms. This mode of gene silencing, called RNA interference (RNAi), acts at the transcriptional level through formation of heterochromatin, and at the post-transcriptional leve,l through mRNA degradation and translational suppression. In all cases, RNAi begins with the processing of endogenous or introduced precursor RNA into micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) 21-25 nucleotides in length by the enzyme Dicer. The central objective of this project is to determine how RNA is recognized and cleaved by Dicer during induction of the RNAi pathway. We aim to understand the structural and biochemical basis for Dicer activity, including its ability to recognize double stranded RNA targets, produce approximately 22- nucleotide duplex RNA products and deliver these products to downstream silencing pathways. Although miRNAs and siRNAs are the indispensable mediators of all RNAi gene silencing processes, the biochemical basis for their generation remains poorly understood. In order for Dicer to produce functional products from long duplex RNA precursors, the enzyme must coordinate two double stranded cleavages over a distance of 60 A. The structural mechanism underlying this coordination is entirely unknown. Well-diffracting crystals are in hand for an intact Dicer enzyme and will be the focus of initial structure determination efforts using X-ray crystallography. In parallel, a series of in vitro and in vivo experiments will be conducted to elucidate mechanisms of RNA recognition and catalysis by different kinds of Dicers. The proposed studies will reveal the molecular "ruler" used to determine siRNA length and may enable the design of pre-siRNAs targeted to specific gene silencing pathways.